Scanning probe microscopy (SPM) is a technique in which a measurement probe is scanned over a sample, which can also be referred to as the measurement object or as the measurement object to be examined, and the topography is determined via a distance-dependent interaction between the measurement probe and the sample. Material contrasts or other sample information can also be obtained. The most prominent examples of this measurement technique are the atomic force microscope (AFM) and the scanning tunneling microscope (STM). Further examples are the scanning near-field microscope (SNOM) and the scanning photon force microscope (SPhM).
In addition to imaging the measurement object, distance spectroscopy is another important examination method in all these techniques. Here, the measurement probe is displaced relative to the sample, in particular in the vertical direction or in any direction in space or in one plane, and the interaction is measured. In the case of atomic force microscopy, this method is used, for example, to measure the forces between molecules, one molecule being bound to the measurement probe and another molecule being bound to the sample. It is also possible to measure intramolecular forces, for example by lowering the measurement probe onto the sample and waiting on bonding. Thereafter, the measurement probe can be removed from the base on which the sample is arranged, and the force can be recorded. Further measurements may also be provided for, and such measurements are in part also carried out by measuring an interaction which is correlated with the distance between two or several points.
Optical methods such as fluorescence microscopy, for example, are able to supply information about the composition of the sample examined, for example by labeling particles with specific fluorescence markers. Furthermore, FRET (fluorescence resonance energy transfer), for example, allows the localization of two labeled molecules with respect to one another.
If SPM is used as the examination method, for example in the distance spectroscopy mode described above, often a displacement of the measurement object is produced which then also has effects on the fluorescence or other optical properties of the sample. In order to be able to optically observe these properties, an observation region of an optical measurement system which is used for the optical examination of the sample, preferably the focus, must spatially overlap with the optically examined section of the measurement object. The measurement object must also be close enough to the optical axis that it can be detected by the optical measurement system, for example by means of a measurement objective. Various scenarios for the displacement of the measurement object brought about by the examination by means of scanning probe microscopy are possible, in which the distance between the measurement object and the measurement objective is changed, as a result of which the measurement object is possibly displaced out of the observation region of the optical measurement system, for example the focusing plane of a measurement objective:                The support for the sample is moved, for example by means of a piezo arrangement, in order to vary the distance between the sample and the measurement probe. If the object to be optically examined, in particular a subsection of the sample, is fixedly connected to the sample, this leads to defocusing.        The measurement probe is moved by means of a piezo arrangement in order to vary the distance between the sample and the measurement probe. If the object to be optically examined is fixedly connected to the measurement probe, this leads to defocusing.        If the measurement object is a part of the sample which is varied or displaced by the acting forces, defocusing will take place regardless of the moving part.        
Since the stretching widths in distance spectroscopy may often be 100 μm or more, defocusing is not acceptable for the further optical examination.